RESUMO
In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca(2+)-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 µM TG or 25 µM 2',5'-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca(2+) and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.
Assuntos
ATPases Transportadoras de Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Bombas de Íon/efeitos dos fármacos , Tapsigargina/farmacologia , Trypanosoma/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/imunologia , Retículo Endoplasmático/enzimologia , Doenças dos Cavalos/parasitologia , Cavalos , Bombas de Íon/metabolismo , Masculino , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Trypanosoma/efeitos dos fármacos , Trypanosoma/fisiologia , Tripanossomíase/parasitologia , Tripanossomíase/veterináriaRESUMO
Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.
Assuntos
Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Trypanosoma vivax/imunologia , Trypanosoma/imunologia , Tripanossomíase Bovina/imunologia , Glicoproteínas Variantes de Superfície de Trypanosoma/imunologia , Animais , Especificidade de Anticorpos , Antígenos de Protozoários/imunologia , Cálcio/metabolismo , Bovinos , Proteínas do Sistema Complemento , Soros Imunes , Trypanosoma/classificação , Trypanosoma/metabolismo , Trypanosoma vivax/metabolismo , Trypanosoma vivax/patogenicidade , Tripanossomíase Bovina/parasitologia , Glicoproteínas Variantes de Superfície de Trypanosoma/isolamento & purificaçãoRESUMO
Tubulin is the predominant phosphoprotein in Trypanosoma cruzi epimastigotes and is phosphorylated by a protein kinase CK2. Interestingly, the presence or absence of divalent cations affected the solubilization of a pool of the parasite tubulin and the CK2 responsible for its phosphorylation. This fraction of tubulin and its kinase co-eluted using phosphocellulose, DEAE-Sepharose and Sephacryl S-300 chromatographies. Anti-alpha tubulin antibodies co-immunoprecipitated both tubulin and the CK2 responsible for its phosphorylation, and anti-CK2 alpha-subunit antibodies immunoprecipitated radioactively labelled alpha and beta tubulin from phosphorylated epimastigote homogenates. Additionally, native polyacrylamide gel electrophoresis of the purified and radioactively labelled fraction containing tubulin and its kinase demonstrated the phosphorylation of a unique band that reacted with both anti-CK2 alpha-subunit and anti-tubulin antibodies. Together, these results establish a strong interaction between a pool of the heterodimeric alpha/beta tubulin and a CK2 in this parasite. Hydrodynamic measurements indicated that the T. cruzi tubulin-CK2 complex is globular with an estimated size of 145.4-147.5 kDa.
Assuntos
Caseína Quinase II/metabolismo , Trypanosoma cruzi/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Anticorpos Antiprotozoários/imunologia , Western Blotting/métodos , Caseína Quinases/metabolismo , Cátions Bivalentes/metabolismo , Cátions Bivalentes/farmacologia , Células Cultivadas , Cromatografia/métodos , Imunoprecipitação/métodos , Fosforilação , Ligação Proteica/fisiologia , Coelhos , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/imunologiaRESUMO
Trypanosoma evansi and Trypanosoma vivax are the most extensively distributed trypanosomes responsible for diseases in livestock. Western blot and indirect immunofluorescence assays revealed a high immunological cross-reaction between these two parasites. An antigen with an apparent molecular mass of 64 kDa (p64), which exhibited cross-reactivity with T. vivax, was purified to homogeneity from a Venezuelan isolate of T. evansi. This antigen is glycosylated, contains a glycosyl-phosphatidylinositol anchor and appeared to be localized through the cell except in the nucleus, indicating that it could primarily be confined to the parasite surface. These results, together with its relative abundance and apparent molecular weight, suggest that p64 probably corresponds to the soluble form of a variable surface glycoprotein from T. evansi. Anti-p64 polyclonal antibodies, raised on mice, recognized a 53 kDa polypeptide band from a Venezuelan isolate of T. vivax on Western blots. Additionally, sera obtained from naturally infected animals also recognized p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, demonstrating that another relevant cross-reacting epitope, different than the inositol-1,2-cyclic phosphate of the cross-reacting determinant, must exist in p64. To date, p64 represents the first antigen isolated and partially characterized from T. evansi.